Differential regulation of B cell development, activation, and death by the src homology 2 domain-containing 5' inositol phosphatase (SHIP)

Abstract

Although the Src homology 2 domain-containing 5' inositol phosphatase (SHIP) is a well-known mediator of inhibitory signals after B cell antigen receptor (BCR) coaggregation with the low affinity Fc receptor, it is not known whether SHIP functions to inhibit signals after stimulation through the BCR alone. Here, we show using gene-ablated mice that SHIP is a crucial regulator of BCR-mediated signaling, B cell activation, and B cell development. We demonstrate a critical role for SHIP in termination of phosphatidylinositol 3,4,5-triphosphate (PI[3,4,5]P(3)) signals that follow BCR aggregation. Consistent with enhanced PI(3,4,5)P(3) signaling, we find that splenic B cells from SHIP-deficient mice display enhanced sensitivity to BCR-mediated induction of the activation markers CD86 and CD69. We further demonstrate that SHIP regulates the rate of B cell development in the bone marrow and spleen, as B cell precursors from SHIP-deficient mice progress more rapidly through the immature and transitional developmental stages. Finally, we observe that SHIP-deficient B cells have increased resistance to BCR-mediated cell death. These results demonstrate a central role for SHIP in regulation of BCR signaling and B cell biology, from signal driven development in the bone marrow and spleen, to activation and death in the periphery.

Figure 1

SHIP terminates PI(3,4,5)P₃ signals generated upon BCR aggregation. (A and B) B cells from SHIP^−/− or control littermates were labeled with ³²P-orthophosphate for 1.5 h. The cells were then stimulated with F(ab′)₂ fragments of anti-IgG (12 μg/ml) for the indicated time and immediately lysed with methanol/chloroform. Phospholipids were extracted, deacylated, and fractionated by HPLC. The fractions containing PI(3,4,5)P₃ (A) and the SHIP breakdown product, PI(3,4)P₂ (B), were quantitated by liquid scintillation. (C) IP₃ (Ins 1,4,5 P3) levels in SHIP^−/− and control B cells after stimulation with F(ab′)₂ anti-IgG (12 μg/ml). (D) Intracellular free calcium levels ([Ca2+]_i) were monitored after F(ab′)₂ anti-IgG (10 μg/ml) stimulation of Indo-1–loaded B cells derived from SHIP^−/− or wild-type littermates.

Figure 2

Reduced numbers of immature/transitional, but increased numbers of mature B cells in the spleens of SHIP^−/− mice. Spleen cells from 7-wk-old SHIP^−/− and SHIP^+/+ littermates were RBC-lysed and directly stained (A) for B220, IgM, 7AAD (via probe), and IgD. Cells were analyzed by flow cytometry, and gated for viable/B220⁺ cells. Early transitional cells in region T1 (mIgM^himIgD^lo) decreased from 16 (control) to 4% of the B cell population in SHIP deficiency, late transitional B cells in region T2 are reduced from 20 to 9%, while the percentage of mature cells in region M (IgM^loIgD^hi) are increased 1.5-fold by SHIP deficiency. To eliminate marginal zone cells from the presumptive T1 population (T1), gated cells (IgM^hiIgD^lo/−) were stained for CD21 (B) to determine the population of marginal zone B cells. To further analyze B cell maturation, cells were stained for B220, and HSA (C). A decrease in the transitional HSA^hi population is apparent in SHIP^−/− B cells.

Figure 2

Reduced numbers of immature/transitional, but increased numbers of mature B cells in the spleens of SHIP^−/− mice. Spleen cells from 7-wk-old SHIP^−/− and SHIP^+/+ littermates were RBC-lysed and directly stained (A) for B220, IgM, 7AAD (via probe), and IgD. Cells were analyzed by flow cytometry, and gated for viable/B220⁺ cells. Early transitional cells in region T1 (mIgM^himIgD^lo) decreased from 16 (control) to 4% of the B cell population in SHIP deficiency, late transitional B cells in region T2 are reduced from 20 to 9%, while the percentage of mature cells in region M (IgM^loIgD^hi) are increased 1.5-fold by SHIP deficiency. To eliminate marginal zone cells from the presumptive T1 population (T1), gated cells (IgM^hiIgD^lo/−) were stained for CD21 (B) to determine the population of marginal zone B cells. To further analyze B cell maturation, cells were stained for B220, and HSA (C). A decrease in the transitional HSA^hi population is apparent in SHIP^−/− B cells.

Figure 2

Reduced numbers of immature/transitional, but increased numbers of mature B cells in the spleens of SHIP^−/− mice. Spleen cells from 7-wk-old SHIP^−/− and SHIP^+/+ littermates were RBC-lysed and directly stained (A) for B220, IgM, 7AAD (via probe), and IgD. Cells were analyzed by flow cytometry, and gated for viable/B220⁺ cells. Early transitional cells in region T1 (mIgM^himIgD^lo) decreased from 16 (control) to 4% of the B cell population in SHIP deficiency, late transitional B cells in region T2 are reduced from 20 to 9%, while the percentage of mature cells in region M (IgM^loIgD^hi) are increased 1.5-fold by SHIP deficiency. To eliminate marginal zone cells from the presumptive T1 population (T1), gated cells (IgM^hiIgD^lo/−) were stained for CD21 (B) to determine the population of marginal zone B cells. To further analyze B cell maturation, cells were stained for B220, and HSA (C). A decrease in the transitional HSA^hi population is apparent in SHIP^−/− B cells.

Figure 3

B cell development in SHIP^−/− mice is accelerated. 6–10-wk-old SHIP^−/− mice and wild-type littermates were treated with 500 rads of radiation, and the kinetics of regeneration of B cells in the spleen was monitored. (A) Time course of autoreconstitution of B220⁺ cells in the spleens of SHIP^+/+ and SHIP^−/− mice. (B) 12 and 14 d after sublethal irradiation, splenocytes from 8-wk-old SHIP^+/+ and SHIP^−/− littermates were harvested and stained for the maturation markers IgM and HSA (live/B220⁺ gate). Data shown represent the mean of three independent experiments.

Figure 3

B cell development in SHIP^−/− mice is accelerated. 6–10-wk-old SHIP^−/− mice and wild-type littermates were treated with 500 rads of radiation, and the kinetics of regeneration of B cells in the spleen was monitored. (A) Time course of autoreconstitution of B220⁺ cells in the spleens of SHIP^+/+ and SHIP^−/− mice. (B) 12 and 14 d after sublethal irradiation, splenocytes from 8-wk-old SHIP^+/+ and SHIP^−/− littermates were harvested and stained for the maturation markers IgM and HSA (live/B220⁺ gate). Data shown represent the mean of three independent experiments.

Figure 4

Increased rate of development of immature (IgM⁺) cells in the bone marrow. 7-wk-old SHIP^−/− mice and wild-type littermates were treated with sublethal irradiation (500 rads) to follow the kinetics of regeneration of bone marrow B cells. Bone marrow was harvested from two femurs at the indicated time points (8 and 9 d) after irradiation. Cells were counted and stained for B220, IgM, mAb 493, and 7AAD (via probe). Data show flow cytometric analysis of viable/B220⁺ cells from the bone marrow of SHIP^+/+ and SHIP^−/− mice 8 d (top) and 9 d (bottom) after sublethal irradiation. 250,000 total cells were counted in each case. 8 d after irradiation, the number of B220⁺ cells in the bone marrow of SHIP^+/+ mice was 5.0 × 10⁵, while SHIP^−/− bone marrow had 4.7 × 10⁵ B220⁺ cells. 9 d after irradiation, the number of B220⁺ cells in the bone marrow of SHIP^+/+ mice was 22 × 10⁵, while SHIP^−/− bone marrow had 26 × 10⁵ B220⁺ cells. Data shown are representative of three replicate experiments

Figure 5

SHIP^−/− B cells display increased sensitivity to BCR-mediated induction of activation marker expression. SHIP^−/− or wild-type B cells were stimulated with the indicated concentration of F(ab′)₂ fragments of anti-IgG. (A) After stimulation for 20 h, cells were analyzed. The mean fluorescence intensity of CD86 expression was measured on live/B220⁺ cells. (B) After a 6-h stimulation, the mean fluorescence intensity of CD69 expression was measured. Data shown represent the mean of triplicate determinants.

Figure 6

SHIP^−/− splenic B cells are resistant to BCR-mediated induction of cell death. Freshly isolated splenic B cells from SHIP^−/− mice or control littermates were incubated at 2.0 × 10⁶ cells/ml for 24 h with the indicated concentrations of F(ab′)₂ fragments of anti-IgG. The percentage of surviving cells was determined 24 h later based on recovery of viable (propidium iodide excluding) cells. Data shown represent the mean of triplicate determinants.